The invention relates to preparation and use of viral coat proteins. In particular, the invention relates to preparations of human papillomavirus L1 protein.
Human papillomaviruses are involved in a variety of disease states, including benign warts and cancer. There has been considerable effort to produce vaccines against human papillomaviruses, especially against types 16 and 18, which are associated with cervical cancer. Papillomaviruses contain two structural proteins that encapsidate the viral minichromosome, L1 and L2. In the virus particle, 72 pentamers of L1 form an outer shell; L2, probably one copy per L1 pentamer, is located on the inside of the L1 shell. The L1 and L2 proteins are therefore important candidates to use as immunogens. Because papillomaviruses cannot be propagated in cell culture, however, recombinant methods must be used to produce papillomavirus proteins.
Bacterial expression systems are generally effective and inexpensive ways to produce large quantities of recombinant proteins, but it has been difficult to produce large quantities of papillomavirus L1 protein in bacterial expression systems. Thus, there is a need in the art for methods of obtaining preparations of human papillomavirus L1 proteins which can be used as immunogens and as diagnostic reagents from bacterial expression systems.
It is an object of the present invention to provide stable preparations of human papillomavirus L1 protein for use as immunogens and as diagnostic tools.
One embodiment of the invention is a composition which comprises a multimer of a human papillomavirus L1 protein and a physiologically compatible carrier. As used herein, the term xe2x80x9cL1 proteinxe2x80x9d refers to a truncated L1 polypeptide that does not include amino terminal residues 1-8 of HPV16 L1 , or the corresponding structural residues of the L1 protein of other HPV subtypes.
As used herein, the term xe2x80x9cmultimerxe2x80x9d refers to more than one L1 monomers; however, the invention is most advantageous where xe2x80x9cmultimerxe2x80x9d refers to 5 L1 monomers (i.e., a pentamer) up to and including 60 monomers (12 pentamers).
As used herein, the term xe2x80x9cstablexe2x80x9d means that the preparation of L1 protein is not proteolytically degraded or denatured by proteases when placed in 10 mM salt, 0.1 mM EDTA at 10xc2x0 C. for 24 hours.
Preferably, the preparation of human papillomavirus L1 protein is also soluble.
As used herein, xe2x80x9csolublexe2x80x9d means that the preparation of L1 protein remains in the supernatant after centrifugation in a table-top centrifuge at 10,000 rpm for 5 minutes.
These and other objects of the invention are provided by one or more of the embodiments which are described below.
Another embodiment of the invention is a method of immunizing a human against a human papillomavirus. A composition which comprises a multimer of a human papillomavirus L1 protein is administered to a human at a dose effective to induce an immune response against the L1 protein in the human. As used herein, xe2x80x9ceffective to induce an immune responsexe2x80x9d refers to the ability to induce an antibody response to the L1 protein or to induce a CTL response to L1 .
Yet another embodiment of the invention is a method of detecting the presence of antibodies against a human papillomavirus in a biological sample. A biological sample is contacted with a multimer of a human papillomavirus L1 protein. Antibodies which bind to the multimer are detected. Detection of antibodies which are bound to the multimer identifies the presence of antibodies against the human papillomavirus in the biological sample.
A further embodiment of the invention is a method of detecting a specific subtype of human papillomavirus in a biological sample. A biological sample is contacted with an antibody which specifically binds to an L1 protein of a specific subtype of human papillomavirus. L1 protein which is bound to the antibody is detected. Detection of L1 protein which is bound to the antibody identifies the presence of the specific subtype of human papillomavirus in the biological sample.
Even another embodiment of the invention is a solid support comprising a multimer of a human papillomavirus L1 protein.
Another embodiment of the invention is a solid support comprising an antibody which specifically binds to an L1 protein of a specific type of human papillomavirus.
Still another embodiment of the invention is a method of producing a soluble multimer of a human papillomavirus L1 protein which is truncated at its amino terminus. A recombinant human papillomavirus L1 protein is expressed in a bacterial host cell. The recombinant human papillomavirus L1 protein comprises amino acids 9-505 as shown in SEQ ID NO: 1. Preferably the L1 protein comprises amino acids 10-505, 11-505, 12-505, and even 13-505 as shown in SEQ ID NO:1. The recombinant human papillomavirus L1 protein is treated to remove bacterial host cell proteins. A soluble multimer of the human papillomavirus L1 protein is thereby formed.
The present invention thus provides the art with a simple and effective method of producing large quantities of human papillomavirus L1 protein. Preparations of human papillomavirus protein can be used, inter alia, as immunogens and diagnostic tools.